Abstract: Tomato fruits infected with Penicillium funiculosum Thom. produced proteins which showed appreciable polygalacturonase activity within eight days. Uninfected tomato fruits showed only traces of polygalacturonase activity. The enzyme was partially purified by a combination of gel filtration and ion-exchange chromatography. Two components with molecular weight estimates of approximately 223,800 daltons and 89,100 daltons were expressed. Only the components of the lighter peak showed polygalacturonase activity. The enzyme showed optimum activity at pH 4.5 and 40°C. It possessed an apparent Km of 0.05 mg mL-1 for the hydrolysis of pectin. Na+ and CaH ions were stimulatory to the activity of the enzyme. EDTA and Hg++ were inhibitory.
Introduction
Tomato fruits are consumed worldwide. Tomato is an important vegetable accounting for approximately 18% of daily consumption of vegetables (Kateria and Mittal, 1984). Tomato fruits contain sugars at varying proportions. These sugars are mainly reducing sugars of approximately 1.3% glucose and 1.5% fructose (Stephens, 1978; Lamb, 1977). They also contain approximately 8% protein, 7% pectic substances and organic acids: mainly citric and malic acids (Crookes and Grierson, 1983). Although the nutritional value of tomato is rather low, the large amount of tomato consumed in any meal makes it quite valuable in standard and special diets (Moresi and Liverotti, 1982).
Fungi are found to be responsible for majority of tomato spoilage during storage (Adejuwon and Olutiola, 2005).
This study shows the degradation of the pectic portions of tomato fruits by Polygalacturonase produced by Penicillium funiculosum Thom. during infection. Studies carried out on the properties of this enzyme are described.
Materials and Methods
Organism and Culture Conditions
The isolate of Penicillium funiculosum Thom. used was obtained from
the culture collection of the Department of Microbiology, Obafemi Awolowo University,
Ile-Ife, Osun state. Nigeria. The organism was routinely grow and maintained
on 1% malt yeast-extract glucose agar slants. Five day old cultures served as
inoculum.
Inoculation of Tomato Fruits
Apparently healthy, ripe tomato (Lycopersicon esculentum Mill.) fruits
were obtained from the Ile-Ife local main market. They were surface sterilized
in 3% sodium hypochlorite for 30 min and thoroughly rinsed with sterile distilled
water. A cork borer (3 mm) was used to remove tissue disc from each tomato fruit
prior to inoculation with tissue discs (3 mm) of 5-day old culture of Penicillium
funiculosum in plates. The holes were sealed with paraffin wax. Tomato fruits
inoculated with sterile malt yeast extract-glucose agar served as controls.
Procedures were carried out in a sterile inoculating chamber. Experimental and
control fruits were placed inside sterile petri dishes with bell jars inverted
over them. The rims of the bell jars were sealed with Vaseline. Incubation was
at room temperature (27°C) The fruits were observed daily for deterioration.
Extraction of Enzyme from Tomato Fruits
Within eight days of incubation, the inoculated tomato fruits had collapsed.
The fruits were weighed, chilled in a refrigerator, homogenized with cold (4°C)
liquid extractant (1:1w/v). The extractant was 0.5 M NaCl in 0.0l M citrate
phosphate buffer (pH 5.0) containing 5 mM sodium azide to prevent microbial
contamination. The homogenate was filtered through four layers of muslin cloth
and further clarified by filtering through filter paper (Whatman No. 1). The
filtrate served as the crude enzyme. The protein content of the filtrate was
determined using the method of Lowry et al. (1951). Polygalacturonase
activity was analyzed using a modified method of Miller (1959).
Preparation of Extract for Fractionation
The crude enzyme homogenate was concentrated to about one seventh of its
original volume in a vacuum rotary evaporator (Quickfit, Rotavapor-R, Buchi,
Switzerland) at 30°C. Frothing was avoided during this process by slow evaporation
under low vacuum pressure (Whitaker et al., 1963).
Fractionation on Sephadex G-25 Column
The column (2.5x40 cm) of Sephadex G-25 (bead size 50-150 μ) was prepared
in a column made up of a glass tube with water jacket supplied by Pharmacia
Fine Chemicals, Uppsala, Sweden. This is as previously described by Olutiola
and Cole (1980). It was equilibrated with 0.0IM citrate phosphate buffer pH
5.0 containing 5 mM NaN3. Fractions were collected (5 mL/tube). Optical
densities of fractions were measured at 280 nm. The fractions were analyzed
for polygalacturonase activity.
Fractionation on Sephadex C-50 Column
Fractions (11-17) from Sephadex G-25 which showed appreciable polygalacturonase
activity were pooled together. Six milliliter of the pooled enzyme was applied
to Sephadex C-50 column (2.5 x 40 cm) (bead size 40-120 μ) with water jacket,
supplied by Pharmacia fine Chemicals, Uppsala, Sweden. Fractions (5 mL/tube)
were eluted with 0.0I M citrate phosphate buffer pH 5.0 containing a gradient
(0.1-0.5 M) NaCl. Optical density of the fractions was measured at 280 nm. Fractions
were analyzed for polygalacturonase activity.
Fractionation on Sephadex G-100 Column
Fractions (9-12) collected form Sephadex C-50 column which showed appreciable
polygalacturonase activity were pooled together. Four milliliter of the mixture
was applied to Sephadex G-100 column (2.5x70 cm) which had earlier been calibrated
with proteins of known molecular weight as previously described by Andrews (1964),
Olutiola and Cole (1976). Optical density of fractions (5 mL/tube) eluted with
0.0l M citrate phosphate buffer pH 5.0 was measured at 280 nm. The fractions
were analyzed for polygalacturonase activity.
| Table 1: | Partial purification of polygalacturonase from tomato fruits infected by Penicillium funiculosum Thom. |
Assay Method
Polygalacturonase activity was analyzed by measuring the reducing sugars
released in the reaction mixtures using a modified dinitro salicylic acid reagent
method of Miller (1959). The substrate, 0.1% (w/v) pectin (Sigma) solution,
was prepared in 0.0l M citrate phosphate buffer pH 5.0.
The reaction mixture was 0.5 mL of enzyme preparation added to 1 mL of the substrate. Incubation was at 35°C for 1h. The reaction mixture was terminated by adding 3 mL of dinitro salicylic acid (DNSA) reagent. Controls which initially contained only 1 mL of the substrate were incubated with the experimental tubes at 35°C for 1 h. Three milliliter of dinitro salicylic acid was added to each control tube after which 0.5 mL of enzyme was added. The reducing sugar released in the reaction mixture was then measured. One unit of polygalacturonase activity was defined as the amount of enzyme in 1 mL of the reaction mixture that liberated reducing sugars equivalent to 100 μg galacturonic acid per minute under assay conditions. Specific activity was calculated as enzyme unit per mg protein.
Results
Within eight days, tomato fruits infected with Penicillium funiculosum Thom., incubated at room temperature (27°C), had collapsed and extensively overgrown by mycelia and greenish spores. Extracts of the infected fruits exhibited appreciable amount of polygalacturonase activity. Uninfected fruits showed traces of polygalacturonase activity. Fractionation of the concentrated extract on Sephadex G-25 column gave two peaks of absorption. Only the components of the first peak showed polygalacturonase activity. Fractionation of the first peak on Sephadex C-50 column gave two peaks of absorption. Components of the first peak showed polygalacturonase activity. Further fractionation of the components of this peak on Sephadex G-100 column gave two peaks of absorption with molecular weight estimates of 223,800 daltons and 89,100 daltons. Only the components of the lighter peak showed appreciable polygalacturonase activity. The purification steps are shown in Table 1.
Properties of the partially purified polygalacturonase were investigated. Using 0.0IM citrate phosphate buffer with pH ranges of 3.0-7.0, optimum activity was observed at pH 4.5. With a temperature range of 20 to 50°C, optimum activity was observed at 40°C. The polygalacturonase was stimulated by Na+ and CaH ions. Optimum activity was observed at 20 mM concentrations of Na+ ions and CaHions. With different concentrations of pectin as substrate (ranges of 0.05-6 mg mL-1), optimum activity was recorded over a range of 4-6 mg mL-1. The rate of enzyme reaction seemed to follow the Michalis-Menten Kinetics. From the Lineweaver Burk Plot, the apparent Km for the hydrolysis of pectin was approximately 0.05 mg mL-1. The polygalacturonase was inhibited by EDTA and HgH ions. There was total inhibition at 6 and 4 mM concentrations, respectively.
Discussion
Extensive deterioration and fruit rot occurred within a few days of infection of tomato fruits by Penicillium funiculosum Thom. Similar reports have been made of this fungi on pineapple fruits (Lim and Rohrbach, 1980). Polygalacturonase was detected in the extracts of infected tomato fruits. Penicillium sclerotigenum has been found to produce this enzyme in synthetic liquid medium with pectic substance as sole corbon source (Olutiola, 1982).
The enzyme showed optimum activity at pH 4.5. A similar result has been documented by Perez-artes and Tena (1989) for Fusarium oxysporum. The enzyme was affected by temperature changes. Optimum activity was observed at 40°C. However, Agarwal et al. (1979) reported a lower optimum temp of the range 30-35°C for polygalacturonase produced by Curvularia lunata and Colletotrichum dematium. The enzyme was able to degrade pectin with optimum activity at a range of 4-6 mg mL-1 concentrations. A similar report has been described by Aymerig et al. (1989). Activity of the enzyme was stimulated by Na+ and CaH ions. Popoola (1987) made an almost similar report on polygalacturonase produced by Aspergillus niger. causing the black mould rot of yam.
Activity of the enzyme was inhibited by EDTA and HgH ions. Reports made by Yoichi et al. (1993) indicate that polygalacturonase from strawberry fruits are inhibited similarly. The results of this study show that polygalacturonase is produced by Penicillium funiculosum Thom. during infection to hydrolyse the pectic portion of tomato fruits into simple soluble forms such as polygalacturonic acid. These are then subsequently absorbed and metabolized by this phytopathogen for growth.